Utilizing a combined biochemical and morphological approach it is the objective of this proposal to define the molecular alterations that have occurred in the plasma membrane of erythrocytes from patients with sickle cell anemia. Because therapy which focuses on the S-hemoglobin to prevent the cells from sickling generally has not been successful in relieving the vaso-occlusive painful crises, we propose to examine the membranes of the erythrocyte with S-hemoglobin; our ultimate goal being successful therapy based on knowledge of the membrane defects. To define the membrane proteins of normal, sickled and irreversibly sickled erythrocytes, one and then two dimensional polyacrylamide gel electrophoresis will be utilized. Enzymatic digestions will then be used to determine whether the components which differ between normal and sickle cells are on the inner or outer surface of the membrane. Use of reversible disulfide cross-linking agents and gel electrophoresis to determine which proteins are closely apposed in the sickled erythrocyte membrane will provide a precise molecular map of the protein interactions in the membrane, a map which can be compared with the normal erythrocytes. At a morphological level, the outer surface of the membranes of normal and sickled cells can be compared with respect to the number and distribution of concanavalin A binding sites, using freeze-etch electron microscopy and scanning electron microscopy. Differences in number and distribution of exposed surface anionic sites will be determined by incubating cells in cationized ferritin and detecting the bound ferritin by transmission electron microscopy. Possible differences within the membranes will be detected by a freeze cleave electron microscopic study which will reveal any alterations in size, number or distribution of intramembranous particles. At the inner surface of the membrane, differences in the actin-spectrin complex may be revealed by heavy meromyosin treatment of ghosts of normal and sickled cells followed by transmission electron microscopy to determine the extent of actin filament formation.